Field Assessment of a Model Tuberculosis Outbreak Response Plan for Low-Incidence Areas

Background: For a regional project in four low-incidence states, we designed a customizable tuberculosis outbreak response plan. Prior to dissemination of the plan, a tuberculosis outbreak occurred, presenting an opportunity to perform a field assessment of the plan. The purpose of the assessment was to ensure that the plan included essential elements to help public health professionals recognize and respond to outbreaks. Methods: We designed a semi-structured questionnaire and interviewed all key stakeholders involved in the response. We used common themes to assess validity of and identify gaps in the plan. A subset of participants provided structured feedback on the plan. Results: We interviewed 11 public health and six community stakeholders. The assessment demonstrated that (1) almost all of the main response activities were reflected in the plan; (2) the plan added value by providing a definition of a tuberculosis outbreak and guidelines for communication and evaluation. These were areas that lacked written protocols during the actual outbreak response; and (3) basic education about tuberculosis and the interpretation and use of genotyping data were important needs. Stakeholders also suggested adding to the plan questions for evaluation and a section for specific steps to take when an outbreak is suspected. Conclusion: An interactive field assessment of a programmatic tool revealed the value of a systematic outbreak response plan with a standard definition of a tuberculosis outbreak, guidelines for communication and evaluation, and response steps. The assessment highlighted the importance of education and training for tuberculosis in low-incidence areas

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

BACKGROUND: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. RESULTS: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region. CONCLUSIONS: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment

Mass Die-Off of Saiga Antelopes, Kazakhstan, 2015

In 2015, a mass die-off of ≈200,000 saiga antelope in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes

Diabetes in Danish Bank Voles (M. glareolus): Survivorship, Influence on Weight, and Evaluation of Polydipsia as a Screening Tool for Hyperglycaemia

BACKGROUND: Previous studies have concluded that the development of polydipsia (PD, a daily water intake ≥ 21 ml) among captive Danish bank voles, is associated with the development of a type 1 diabetes (T1D), based on findings of hyperglycaemia, glucosuria, ketonuria/-emia, lipemia, destroyed beta cells, and presence of autoantibodies against GAD65, IA-2, and insulin. AIM AND METHODS: We retrospectively analysed data from two separate colonies of Danish bank voles in order to 1) estimate survivorship after onset of PD, 2) evaluate whether the weight of PD voles differed from non-PD voles, and, 3), evaluate a state of PD as a practical and non-invasive tool to screen for voles with a high probability of hypeglycaemia. In addition, we discuss regional differences related to the development of diabetes in Scandinavian bank voles and the relevance of the Ljungan virus as proposed etiological agent. RESULTS: We found that median survival after onset of PD is at least 91 days (lower/upper quartiles = 57/134 days) with a maximum recording of at least 404 days survivorship. The development of PD did not influence the weight of Danish bank voles. The measures of accuracy when using PD as predictor of hyperglycaemia, i.e. sensitivity, specificity, positive predictive value, and negative predictive value, equalled 69%, 97%, 89%, and 89%, respectively. CONCLUSION: The relatively long survival of Danish PD bank voles suggests potentials for this model in future studies of the long-term complications of diabetes, of which some observations are mentioned. Data also indicates that diabetes in Danish bank is not associated with a higher body weight. Finally, the method of using measurements of daily water intake to screen for voles with a high probability of hyperglycaemia constitutes a considerable refinement when compared to the usual, invasive, methods

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

CALCULATION OF CHEMICAL EQUILIBRIUM IN THE INTERSTELLAR CLOUD

In order to calculate the molecular concentrations in the interstellar cloud and to obtain heir spatial distribution we have made a computational code to solve the set of equations for chemical equilibrium and equations of charge and atom balance. Now 109 molecules and 1070 chemical reactions are included. Besides, the gas phase reactions with neutrals and ions, formation of molecular hydrogen on grains and the ionization and dissociation due to cosmic rays and UV radiation were taken into account. In order to calculate the photodissociation rates of H2 and CO molecules we take into consideration: i) extinction due to H2 Lyman and Werner series bands, ii) extinction due to atomic H Lyman series lines - up to n=1 -> n=51, iii) extinction due to 33 CO bands and dust continuum extinction. Only UV continuum for photodissociation of all other species was taken into account